Studies have been continued to understand the regulation and role of neurofilament phosphorylation in the nervous system. Axonal neurofilament-rich preparation from squid giant axon contains second messenger-independent protein kinases that phosphorylate high molecular weight >400 and 220 kDa squid neurofilament protein subunits, as well as exogenous substrates. Two major kinase activities were. separated and characterized in this preparation. One of these strongly phosphorylated kemptide and was inhibited by the selective inhibitors of cAMP-dependent kinase, wiptide. The second kinase activity effectively phosphorylated alpha-casein and was not inhibited by wiptide and heparin. The alpha-casein phosphorylating activity was the principal activity responsible for neurofilament protein phosphorylation and was not inhibited by various kinase inhibitors. A newly synthesized isoquinoline derivative (CKI-7) that specifically inhibited purified casein kinase-I was the effective inhibitor of the axonal neurofilament protein kinase. The physical, biochemical and pharmacological studies indicated that the major kinase activity associated with axonal neurofilaments is like a casein kinase 1. The mammalian neurofilament associated kinases appear to be more complex. There was only a partial inhibition of bovine neurofilament kinase activity by casein kinase I inhibitor and the tryptic peptide maps of neurofilament protein subunit (NF-M) after its phosphorylation by bovine neurofilament kinase showed some similarities when phosphorylated by purified casein kinase I or casein kinase 11. The cAMP-dependent kinase phosphorylated distinct peptides on NF-M. We have determined the sequence of a squid neuronal intermediate filament protein, approximately 59 kDa, using a cDNA library made from squid optic lobe. This protein is specific to neural tissue, and is present in the axoplasm of the giant axon. Structurally, its rod domain possesses features common to mammalian Type IV (neurofilament) and Type III (vimentin, GFAP) intermediate filament proteins as well as Lamins. By highlighting those sequences of NF-proteins that are conserved between these two animals, which diverged around 570 million years ago, the sequence of this mRNA will help elucidate functionally important domains in NF-proteins.